The “multi-functional” Ca²⁺ and calmodulin-dependent protein kinase, type II (CaMK-II) is an evolutionarily conserved protein. It has been found as a single gene in the horseshoe crab, marine sponge, sea urchin, nematode, and fruit fly, whereas most vertebrates possess four genes (α, β, γ, and δ). Species from fruit flies to humans encode alternative splice variants which are differentially targeted to phosphorylate diverse downstream targets of Ca²⁺ signaling. By comparing known CaMK-II protein and nucleotide sequences, we have now provided evidence for the evolutionary relatedness of CaMK-IIs. Parsimony analyses unambiguously indicate that the four vertebrate CaMK-II genes arose via repeated duplications. Nucleotide phylogenies show consistent but moderate support for the placement of the vertebrate δ CaMK-II as the earliest diverging vertebrate gene. δ CaMK-II is the only gene with both central and C-terminal variable domains and has three to four times more intronic sequence than the other three genes. β and γ CaMK-II genes show strong sequence similarity and have comparable exon and intron organization and utilization. α CaMK-II is absent from amphibians (Xenopus laevis) and has the most restricted tissue specificity in mammals, whereas β, γ, and δ CaMK-IIs are expressed in most tissues. All 38 known mammalian CaMK-II splice variants were compiled with their tissue specificity and exon usage. Some of these variants use alternative 5′ and 3′ donors within a single exon as well as alternative promoters. These findings serve as an important benchmark for future phylogenetic, developmental, or biochemical studies on this important, conserved, and highly regulated gene family.